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J Virol. 2010 March; 84(6): 2732–2739.
Published online 2010 January 6. doi:  10.1128/JVI.01561-09
PMCID: PMC2826077

In Vitro Synthesis of Sindbis Virus Genomic and Subgenomic RNAs: Influence of nsP4 Mutations and Nucleoside Triphosphate Concentrations [down-pointing small open triangle]

Abstract

Two positive-strand mRNAs are made in Sindbis virus-infected cells, the genomic (G) RNA and the subgenomic (SG) RNA. In mosquito cells infected with wild-type (wt) Sindbis virus, the latter is made in excess over the former; however, in cells infected with SVpzf or SVcpc more G RNA is made than SG RNA. Use was made of in vitro systems to investigate the effects of the SVpzf and SVcpc mutations on the synthesis of SG and G RNAs. Our findings indicate that under standard reaction conditions, the SG/G RNA ratio in vitro reflects the ratio of SG to G RNA made in infected mosquito cells. We observed further that the RNA patterns seen in vitro are affected not only by the SVpzf and SVcpc mutations but also by the nucleoside triphosphate concentrations in the reaction mixtures and that introduction of these mutations into nsP4 and the promoter/template change the relative amounts of SG and G RNAs that are made, likely through the choice of promoter. We conclude that with respect to the SVpzf and SVcpc mutations, it is mainly the nucleotide changes in the SG promoter, not the amino acid changes in nsP4, that determine the SG/G RNA ratio that results. Further, it was observed that the SVpzf mutations enhance the in vitro synthesis of SG RNA at the lowest concentrations of UTP/CTP and that the single SVcpc mutation enhances the synthesis of G RNA at the lowest concentrations of CTP tested. We also identified three Arg residues in nsP4, R545, R546, and R547, that are needed for the synthesis of G RNA but not SG RNA.

Sindbis virus (family Togaviridae, genus Alphavirus) is one of the simplest enveloped RNA viruses (see references 10 and 25 for reviews). It contains a single-stranded positive-sense RNA genome, about 11,700 nucleotides in length, which encodes four nonstructural (ns) proteins, nsP1, nsP2, nsP3, and nsP4. These four proteins exist in infected cells as a multiprotein complex (2, 18) which serves to synthesize and modify viral RNA. Specific biochemical roles have been associated with three of the proteins in this complex. nsP1 is involved in the capping and methylation of the viral mRNAs (1, 22). nsP2 has a protease domain (7) which functions to cleave the polyproteins P123 and P1234, from which the individual ns proteins are derived; it also has an RNA helicase domain (4). nsP4 is the viral RNA polymerase. The function of nsP3, the least conserved of the viral ns proteins and the only one that is phosphorylated (14), is not clearly understood.

Following viral entry into the host cell and synthesis of the four ns proteins, a full-length negative-strand copy of the viral genome is made; this negative-strand RNA, in turn, serves as the template for two positive-sense viral RNAs, the genomic (G) RNA and a subgenomic (SG) RNA. The latter is about 4,100 nucleotides (nt) in length and encodes the structural proteins of the virus; its sequence is identical to the 3′-terminal 4,100 nucleotides of the G RNA. The negative-strand RNA contains two promoter regions, one is located at the 3′ end of the negative strand and controls the synthesis of G RNA (3, 19), and the second, an internal promoter, controls the synthesis of SG RNA. Full activity of the SG promoter is observed when the negative-strand RNA sequence corresponding to the region from position −98 to +14, (where +1 [nt 7598] represents the SG RNA initiation site) is present (28). Since the synthesis of the G RNA begins at or near the 3′ terminus of the negative-strand RNA and the synthesis of the SG begins internally, it is likely that the initiation mechanisms for the syntheses of these two RNAs are different. Given that the multiprotein complex containing the four ns proteins is responsible for both replication of the viral genome and transcription of the SG RNA, it will be referred to as the replicase/transcriptase (R/T) complex.

In general, cells infected with wt alphaviruses make SG RNA in amounts equal to or in excess of G RNA (8, 15, 20). However, viral mutants have been described which make less SG RNA than G RNA. In many cases this reversal of the SG/G RNA ratio is associated with a decreased virus yield, sometimes in a host-dependent manner (9). Mutants deficient in the synthesis of SG RNA fall into three classes: (i) promoter mutants, i.e., mutants with a nucleotide change in the promoter for SG RNA (5, 8, 20); (ii) nsP2 mutants, in which the mutation results in a decreased activity of the nsP2 protease (26); (iii) a single mutant with a mutation in nsP3 (11). How the mutation in nsP3 decreases the synthesis of SG RNA is not known.

We have described two mutants of Sindbis virus, SVpzf and SVcpc, which show an altered pattern of RNA synthesis in that they make less SG RNA than G RNA (15, 20). Both SVpzf and SVcpc are nsP4 mutants. However, since the coding sequence for the C-terminal portion of nsP4 overlaps with the SG promoter (25), and since the SVcpc mutation and two of the SVpzf mutations lie within the sequence that constitutes the full-length SG promoter (28), it is not clear whether this reversal of the SG/G RNA ratio is due to the changes in nsP4, to the change in the sequence of the SG promoter, or to both (Table (Table11 shows the SVpzf and SVcpc mutations and amino acid changes in nsP4).

TABLE 1.
Mutations in SVpzf and SVcpc

SVpzf was selected on the basis of its resistance to pyrazofurin (PZF), a nucleoside analog which blocks an early step in the pyrimidine biosynthetic pathway and thus lowers the levels of both UTP and CTP (21). SVcpc was selected on the basis of its resistance to cyclopentenylcytosine (CPC), a compound which inhibits CTP synthase, thereby lowering the level only of CTP (16). Thus, SVpzf is able to replicate in mosquito cells with low levels of UTP/CTP and SVcpc is able to replicate in cells with low levels of CTP.

Earlier we had suggested that SVpzf is able to replicate in PZF-treated mosquito cells, i.e., in cells with low levels of UTP/CTP, because the RNA polymerase which it encodes has an increased affinity for UTP and CTP, enabling it to make viral RNA in the face of low levels of these two substrates (20, 21). While this increased affinity for UTP/CTP is beneficial for the virus when concentrations of UTP/CTP are limiting, this increased affinity may have detrimental effects when SVpzf replicates in cells with normal or increased levels of UTP/CTP (15). This would explain why treatment of SVpzf-infected mosquito cells with PZF increases the virus yield, and why the PZF effect is reversed by addition of uridine (15, 21). It does not explain, however, how PZF changes the pattern of SG versus G RNA synthesis in SVpzf-infected cells, i.e., from an SG/G ratio of <1 to a ratio of >1 (15). Similar considerations would explain why SVcpc is able to replicate in CPC-treated cells, i.e., in cells with low levels of CTP, and why addition of uridine or cytidine (both of which increase UTP/CTP levels) decreases virus yield and viral RNA synthesis in these cells. In the case of SVcpc, we have shown that the R/T complex which it encodes has a lower Km for CTP (but not for UTP) than does that of SVstd (18).

We have described in vitro systems for the synthesis of SV G and SG RNAs (17, 19). The critical elements in these systems are (i) a P15 fraction obtained from BSC40 cells infected with recombinant vaccinia virions expressing the four SV ns proteins and (ii) a negative-strand RNA which serves as a promoter/template (P/T) and which contains either just the promoter for the synthesis of SG RNA or promoters for the synthesis of both SG and G RNA.

Recently we used these systems to compare the in vitro synthesis of G and SG RNAs as a function of the concentration of UTP/CTP, using in one case an SVwt system (both nsP4 and the P/T contained wt sequences) and in the second case an SVpzf system (nsP4 contained the three SVpzf amino acid changes and the P/T contained the SVpzf mutations at the −5 and the −55 positions of the SG promoter) (15). With the SVwt system, at concentrations of UTP/CTP up to 1 mM, only small amounts of RNA were made, but when the UTP/CTP concentrations were raised to 1.5 mM or higher, the amount of RNA made increased sharply. At all concentrations of UTP/CTP, SG RNA was made in excess of G RNA in accordance with what we observe in cells infected with SVwt.

The results with the SVpzf system were quite different. More RNA was made at low concentrations of UTP/CTP (0.2 to 0.5 mM) than in the SVstd system, and at these low concentrations of substrate, more SG RNA was made than G RNA. However, when the concentrations of UTP/CTP were raised to 1.5 mM, the synthesis of SG RNA declined, and the synthesis of G RNA increased. Thus, with the SVpzf system, only at concentrations of UTP/CTP of 1.5 mM or greater did the results in vitro reflect what was observed in SVpzf-infected mosquito cells.

We have now extended our experiments on the influence of nucleoside triphosphate (NTP) concentrations on the in vitro synthesis of Sindbis virus G and SG RNA. We describe here how the patterns of viral RNA synthesis (the ratios of SG/G RNA that are made) are influenced not only by the SVpzf and SVcpc mutations present in nsP4 and the SG promoter but also by the concentrations of ATP, UTP/CTP, and CTP alone. We also made use of the in vitro systems to address the question of whether it is the amino acid changes in nsP4 or the nucleotide changes in the SG P/T that are responsible for the altered patterns of RNA synthesis associated with the SVpzf and SVcpc mutations. Additionally, we have identified three arginine residues in nsP4 which are required for the synthesis of G RNA but not SG RNA.

MATERIALS AND METHODS

Cells and viruses.

BSC40 cells were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. Recombinant vaccinia viruses encoding Sindbis virus P123, nsP4, or T7 polymerase (VTF7-3) were kindly provided by Charles M. Rice and Richard Hardy and were propagated in BSC40 cells as described previously (12). The Sindbis virus P123 we used contained the N614D change in nsP2 that results in more rapid processing of P123 (13, 24) than is observed with the wt P123. The mutations in nsP4 were introduced as described by Li and Stollar (18). The various nsPs were expressed from a T7 promoter.

Expression and preparation of recombinant Sindbis virus nonstructural proteins.

BSC40 monolayers in T75 tissue culture flasks were infected with three different recombinant vaccinia virus vectors; these encode the SV nonstructural polyprotein P123 (the polyprotein precursor of nsP1, nsP2, and nsP3), SV nsP4, and T7 RNA polymerase, each at a multiplicity of infection of 1 PFU/cell. Twenty to 24 h after infection, the P15 fraction was prepared according to the methods of Lemm et al. (12). The P15 pellet isolated from one T75 flask was resuspended in 50 μl of storage buffer (10 mM Tris-Cl [pH 7.8], 10 mM NaCl, 15% glycerol) and used as the source of the Sindbis virus R/T complex.

In vitro synthesis of SV RNA.

The reaction mixture for the in vitro synthesis of SV RNA (25 μl) contained 5 μl of 5× reaction buffer (200 mM Tris-HCl, pH 7.9, 30 mM MgCl2, and 50 mM NaCl), 10 mM dithithreitol, 40 units of RNase inhibitor (Promega), 2 μg of negative-strand RNA, which served as the promoter/template, and 12.2 μl (4.7 μg of protein/μl) of P15 extract prepared from BSC40 cells infected with recombinant vaccinia virus expressing the T7 RNA polymerase, the SV polyprotein, P123, and the SV nsP4 (either the wt or one of the mutant forms, as indicated). Standard reaction mixtures contained 3 mM ATP, 2 mM UTP, 2 mM CTP, and 0.5 mM GTP. [32P]GTP (800 Ci/mM; 10 μCi/μl) was included to label the transcripts. Concentrations of ATP, CTP, and UTP were, however, varied, as indicated for the different experiments. Incubation was at 37°C for 1 h. RNA was extracted using phenol-chloroform and electrophoresed on a 1% denaturing agarose gel containing 2.2 M formaldehyde. The gel was transferred to a Genescreen Plus hybridization transfer membrane (Perkin-Elmer); the membrane was then exposed to a storage phosphor screen and scanned as described previously (15). The intensities of the bands were quantitated using ImageQuant software.

The negative-strand RNA P/T for making SG RNA was as described by Li and Stollar (17), and the P/T for making both SG and G RNA was as described by Li et al. (15). Viral RNA synthesis in vitro was carried out with a wt system (nsP4 and P/Ts contained no mutations), an SVpzf system (nsP4 contained the three SVpzf mutations but the negative-strand P/T contained only the SVpzf mutations at positions −55 [nt 7543] and −5 [nt 7593]), and an SVcpc system (nsP4 and the P/T contained the single SVcpc mutation). As well, mixed or hybrid systems were used which contained either a wt nsP4 and a mutant P/T (SVpzf or SVcpc) or a mutant nsP4 (SVpzf or SVcpc) and a wt P/T.

RESULTS

As described in the introduction, the in vitro synthesis of viral RNAs made by the SVwt and SVpzf systems showed marked differences when RNA synthesis was titrated as a function of the concentration of UTP/CTP. As a next step, we wished to examine RNA synthesis as a function of the concentrations of ATP and in doing so to use not only the SVwt and SVpzf systems but also mixed systems containing mutant nsP4 and wt P/T or the reverse.

Influence of ATP concentration on the in vitro synthesis of Sindbis virus RNAs.

Figure Figure11 shows how the ATP concentration affects in vitro viral RNA synthesis in a system making both SG and G RNA. With the wt system, SG RNA was always made in excess of G RNA, and maximum synthesis of SG and G RNAs was achieved with 4 mM ATP (Fig. (Fig.1A).1A). Increasing the ATP to as high as 8 mM had no further effect. The results with the SVpzf system were strikingly different. Synthesis of G RNA increased as the ATP concentration was raised from 1 to 3 mM but then steadily declined as the ATP concentration was further increased. In contrast, there was a steady increase in the amount of SG RNA made as the ATP concentration was increased from 1 to 8 mM. Thus, in the SVpzf system, whether more SG RNA or more G RNA was made depended on the ATP concentration.

FIG. 1.
In vitro synthesis of SG and G RNAs as influenced by the concentration of ATP and the SVpzf and SVcpc mutations. P15 fractions were prepared as described in Materials and Methods and contained, as indicated, either wt nsP4, nsP4 with the three SVpzf amino ...

In another experiment (Fig. (Fig.1B)1B) we observed that the SVcpc pattern closely resembled the SVpzf pattern. G RNA synthesis peaked at an ATP concentration of 3 mM in both systems and then steadily declined as the ATP concentration was increased. SG RNA synthesis increased steadily with increasing concentrations of ATP up to 7 to 8 mM. To determine which was more critical to the aberrant RNA pattern seen with the SVpzf and SVcpc systems, the mutant nsP4 or the mutant P/T, we ran reactions with mixed systems containing wt nsP4 and mutant P/T or mutant nsP4 and wt P/T. Figures 1C and D show that with the mixed systems, when the P/T was wt and nsP4 was mutant, the RNA pattern was like that of the wt system (Fig. (Fig.1A).1A). SG RNA was always made in excess of G RNA, independent of the ATP concentration, and synthesis of SG RNA and G RNA reached a maximum at 3 mM ATP and then remained level even as the ATP concentration was increased.

When the components were reversed, i.e., when wt nsP4 was incubated with mutant P/T, the RNA pattern resembled the patterns observed with the SVpzf system or the SVcpc system (Fig. (Fig.2B).2B). One notable difference, however, was that in the mixed systems with mutant P/T and wt nsP4, the synthesis of G RNA did not require 3 mM ATP for maximum activity; on the contrary, G RNA synthesis was maximum even at 1 mM ATP. It stayed level up to 2 to 3 mM ATP and then steadily decreased as the ATP concentration was raised further. Thus, the aberrant patterns seen with both the SVpzf and SVcpc systems are due mainly to the mutant P/Ts, rather than to the mutant nsP4s. As with the SVpzf and SVcpc systems, in the mixed systems with mutant P/T, the ratio of SG/G RNA made varied with the ATP concentration.

FIG. 2.
In vitro synthesis of SG and G RNAs as influenced by the concentrations of UTP/CTP and by the SVpzf and SVcpc mutations. The P15 fractions (wt and mutant) and the P/Ts (wt and mutant) were as described for Fig. Fig.1.1. The nsP4 and the P/T used ...

We also carried out similar experiments in an in vitro system that made only the SG RNA. The results (not shown) were similar to those shown for the SG RNAs in Fig. Fig.11.

Influence of UTP/CTP concentrations on in vitro synthesis of Sindbis virus RNAs.

As shown in Fig. Fig.2A,2A, when a wt system was used, SG RNA synthesis was poor when the concentrations of UTP/CTP were 0.2 to 1.0 mM but increased markedly when the UTP/CTP concentrations were raised to 1.5 to 2.0 mM or higher. With the SVpzf system, maximum SG RNA synthesis was achieved even with 0.2 mM UTP/CTP but declined steadily as the concentrations of UTP/CTP were increased to 1.5 mM or higher. It is noteworthy that when the concentrations of UTP/CTP were low, e.g., 0.2 mM, the SVpzf system was much more efficient in making SG RNA than the wt system. Turning to the mixed systems, it was again clear that it was the P/T, not nsP4, that determined the pattern of SG RNA synthesis as a function of the UTP/CTP concentrations (Fig. (Fig.2B).2B). When we used SVpzf P/T and wt nsP4 the RNA pattern was very similar to that seen with the SVpzf system, and when we used wt P/T and SVpzf nsP4, the pattern resembled that seen with the wt system.

We recently described the effects of varying the UTP/CTP concentrations in a system making both SG and G RNA by using the wt system or the SVpzf system (see Fig. Fig.44 in the report of Li et al. [15]). The patterns with the SVwt system were quite straightforward. SG RNA was always made in excess of G RNA, and maximum amounts of RNA were made at 1.5 to 2.0 mM UTP/CTP. With the SVpzf system, at the lowest concentrations of UTP/CTP tested, SG RNA was made in excess of G RNA; however, as the concentrations of UTP/CTP were increased, synthesis of SG RNA declined. In contrast, synthesis of G RNA was maximal with 1.5 mM UTP/CTP and did not decline as the concentrations of UTP and CTP were increased. Thus, at UTP/CTP concentrations of 1.5 mM or higher, G RNA was made in excess of SG RNA. Figure Figure2C2C shows that in a mixed system containing SVpzf P/T and wt nsP4, the RNA pattern as a function of the UTP/CTP concentrations was like that of the SVpzf system; when the reaction mixture contained wt P/T and SVpzf nsP4, the RNA pattern resembled that seen with the SVwt system in that SG RNA was always made in greater amounts than G RNA (15). However, in contrast to the wt system, synthesis of SG RNA was fairly robust even at the lowest UTP/CTP concentrations (0.2 to 0.5 mM).

FIG. 4.
Effect of mutations in nsP4 on the synthesis of G RNA in vitro and on binding of the viral replicase/transcriptase to the G promoter. (A) Amino acid sequence of nsP4 from positions 526 to 547. The underlined sequence was found earlier to bind to the G ...

Next we assessed the effect of the SVcpc mutation on in vitro viral RNA synthesis. Since this mutant was selected for by serial passage in cells with low levels of only CTP, in the following experiments the RNA patterns were examined as a function only of the CTP concentration. Figure Figure3A3A compares the synthesis of SG RNA by a wt system and an SVcpc system. The RNA pattern seen with the wt system is similar to that shown in Fig. Fig.1A1A and and2A.2A. With the SVcpc system, synthesis of SG RNA was much less robust, and varied little as the CTP concentration was raised. With a mixed system containing the wt P/T and SVcpc nsP4 (Fig. (Fig.3B),3B), the pattern was similar to that of the wt system (Fig. (Fig.3A),3A), but with a mixed system containing the SVcpc P/T and wt nsP4, as with the SVcpc system (Fig. (Fig.3A),3A), SG RNA synthesis showed little variation as the CTP concentration was increased from 0.5 to 5 mM.

FIG. 3.
In vitro synthesis of SG and G RNAs as a function of the concentration of CTP. The P15 fractions (wt and mutant) and the P/Ts (wt and mutant) were as described for the preceding figures. The nsP4 and the P/T used in each of the experiments are indicated. ...

We next examined the effect of varying the CTP concentration in systems making both SG and G RNA. With the wt system, synthesis of both SG and G RNAs was optimal with 2.0 to 3.0 mM CTP and did not increase further as the CTP concentration was increased (Fig. (Fig.3C).3C). At all concentrations of CTP, more SG RNA was made than G RNA. The pattern with the SVcpc system differed significantly in that at all concentrations of CTP more G RNA was made than SG RNA. It is also notable that even with only 0.2 mM CTP, the lowest concentration tested, synthesis of G RNA was maximal and changed very little until the CTP concentration was increased to 4 to 5 mM, at which point it decreased (Fig. (Fig.3C).3C). In another experiment with the SVcpc system (data not shown), when the CTP concentration was raised to 6 or 7 mM, the level of G RNA synthesis fell even more. Compared to G RNA synthesis, SG RNA synthesis varied little as the CTP concentration was increased.

When mixed systems were tested, as was observed in the other experiments described above, it was the P/T which was the critical factor in determining the pattern of RNA synthesis (Fig. (Fig.3D).3D). Thus, in a reaction mixture with wt P/T and SVcpc nsP4, the RNA pattern was very similar to that seen with the wt system (Fig. (Fig.3C).3C). In contrast, when SVcpc P/T was used together with SVwt nsP4, the pattern was like that of the SVcpc system; G RNA was made in excess of SG RNA at all concentrations of CTP, and synthesis of G RNA was robust even at 0.2 mM CTP but declined as the CTP concentration was raised to 5 mM.

Three arginine residues in nsP4 that are critical for synthesis of Sindbis virus G RNA.

The amino acid changes in nsP4 encoded by SVpzf and SVcpc are shown in Table Table1.1. In addition to these changes, it would be expected that the synthesis of SG and G RNAs are influenced also by the amino acid residues in the regions of nsP4 that bind to the SG and G promoters. Earlier, we reported that when a 45-nt RNA corresponding to the 3′ terminus of the SV negative-strand RNA and labeled at its 5′ end was incubated with the SV R/T complex, it protected an amino acid sequence in nsP4 identified as 531-LGKPLPAD-538 (Fig. (Fig.4A)4A) (19). We suggested that this sequence was important for the recognition of the promoter for the synthesis of G RNA. However, amino acid sequences involved in the recognition of a nucleic acid sequence often contain two or more adjacent basic residues, and there is only one basic amino acid in this sequence, K533. In looking at nearby sequences we noted a 545-RRR-547 sequence and a KR sequence at residues 526 and 527. We therefore considered the possibility that one or more of these basic residues is also important for the recognition of the G promoter by nsP4. Accordingly, we mutated the nsP4 coding sequence so as to change the amino acids at these positions and introduced these mutations into nsP4 expressed by recombinant vaccinia virions; this enabled us to prepare R/T complexes (P15 fractions) from cells expressing wt P123, along with various mutant forms of nsP4.

Figure Figure4B4B shows the results when RNA-synthesizing reactions were set up containing a wt P/T with both an SG and a G promoter and a P15 fraction from cells infected with recombinant vaccinia virions expressing P123 and wt or mutant nsP4. As expected, when wt nsP4 was expressed, both an SG and a G transcript were made. Such was also the case when Lys 526, Arg 527, Lys 530, or Lys 533 was changed to Ala. Thus, the single basic residue Lys 533, which we identified in the nsP4 sequence that recognized the G promoter, was not critical for the synthesis of G RNA. We also changed Ala 537 to Arg, again without effect. In each case SG RNA was made in excess of G RNA. Quite different results were observed when Arg 545, Arg 546, or Arg 547 was changed to Ala. In each case, although SG RNA was made, there was no synthesis of G RNA.

Figure Figure4C4C shows the results of an electrophoretic mobility shift assay (EMSA) in which the same P15 fractions shown in Fig. Fig.4B4B were incubated with a negative-strand RNA containing the G promoter. The same nsP4 mutations which abolished synthesis of G RNA also abolished binding of the R/T complex to the G promoter. We conclude that R545, R546, and R547 are all required for recognition of the G promoter by the R/T complex and that the failure of nsP4 with Arg-to-Ala changes at any of these three sites to bind to the G promoter explains the lack of G RNA synthesis by these three mutant forms of nsP4.

These nsP4 mutations were also introduced into pToto, the infectious clone of Sindbis virus, and RNA transcripts were prepared. Table Table22 shows the yields of virus obtained when these RNA transcripts were transfected into chicken embryo fibroblasts. RNA transcripts from wt pToto and from pToto encoding the K526A, R527A, K530A, K533A, and A537R nsP4 changes all gave rise to infectious virus. In contrast, when the R545A, R546A, or R547A change was introduced into nsP4, the RNA transcripts were not infectious.

TABLE 2.
Titers of progeny Sindbis virus on CEF

DISCUSSION

The studies reported here show that both the SVpzf mutations and the single SVcpc mutation alter the patterns of in vitro viral RNA synthesis but that in both cases the altered patterns are dependent on the concentrations of the NTP substrates (ATP, UTP/CTP, or CTP alone). Because of the overlap of the SG promoter with the coding sequence of nsP4 (25), certain mutations are able to change both the sequence of the SG promoter and amino acids in nsP4. We showed in experiments with mixed systems that the altered patterns of in vitro viral RNA synthesis seen with the mutant systems were due, in the main, to the mutations in the SG promoter, not to the mutations in nsp4. One notable difference, however, between the pure SVpzf and SVcpc systems and the mixed systems containing a mutant P/T and the wt nsP4 was that at the lowest concentrations of ATP, the synthesis of G RNA was even better in the mixed systems than in the pure mutant systems (Fig. 1B, C, and D). This suggests that nsP4 is not completely without influence on the amount of G RNA that is made.

Relatively little work has been done on how the concentrations of the different NTPs influence the replication of cytoplasmic RNA viruses. Nor is much known concerning the concentration of the NTPs in the cytoplasmic compartments where viral RNA synthesis occurs. Our findings show that the NTP concentrations can influence the choice of promoter (SG versus G) and thus alter the patterns of viral RNA synthesis both in vitro and in vivo. In this connection, it has been reported that the activity of promoters regulating rRNA synthesis in Escherichia coli is very sensitive to NTP concentrations, specifically to the concentration of the initiating NTP (23). Further studies of viral RNA synthesis in cell-free systems should be extremely useful for understanding how the balance between the synthesis of SG and G RNA is established in alphavirus-infected cells.

The robust synthesis in the SVcpc system of G RNA with only 0.2 mM CTP, and of SG RNA in the SVpzf system again with only 0.2 mM UTP/CTP, is likely related to the fact that these mutants were selected for the ability to grow in cells with low levels of CTP in the first instance and of UTP/CTP in the second instance. Why, in both cases, RNA synthesis declines as the particular NTP concentration is increased is more difficult to understand. Perhaps an increased affinity of the RNA-dependent RNA polymerase (RDRP) for the substrate, UTP/CTP or just CTP, as the case may be (for example, see reference 16), along with higher than normal concentrations of that same substrate, interferes with the entry of the other NTPs into the substrate entry channel of the RDRP. The inhibition of G RNA synthesis at high concentrations of ATP in both the SVpzf and the SVcpc systems may, however, require a different explanation.

From their studies, Wielgosz et al. (27, 28) concluded that the sequence −40/+14 of the SG promoter is sufficient for maximum activity of this promoter. However, the finding that the SVcpc mutation at position −75 was able to downregulate the synthesis of SG RNA draws attention to the region further upstream of −40. We therefore used an RNA folding pattern to look for secondary structures in the sequence of the negative-strand RNA upstream of the SG RNA initiation site which might bind viral or cellular proteins and regulate the synthesis of SG RNA. As is usually the case, several different structures were generated, but all featured the extensively base-paired secondary structure, representing the −82/−38 negative-strand sequence shown in Fig. Fig.5.5. We have now begun a mutational analysis of this sequence to determine if disruption of specific base pairs in this structure will affect the synthesis of SG RNA either in vivo or in vitro.

FIG. 5.
Predicted secondary structure of the negative-strand RNA corresponding to the Sindbis virus genome sequence from nt 7516 to 7560. With reference to the SG promoter, this corresponds to the −82/−38 sequence. The initiation site for SG RNA ...

We have also shown in this report that the three Arg residues at positions 545, 546, and 547 of nsP4, which lie close to the sequence that binds to the G promoter, are needed for the synthesis of G RNA but not for the synthesis of SG RNA. In earlier work we showed that the two Arg residues at positions 331 and 332 of nsP4, which are part of the sequence that recognizes the SG promoter, are required for the synthesis of SG RNA but not G RNA. These findings, taken together, provide further support for the view that there are distinct and separate sites on nsP4 for the recognition of the SG and G promoters (6). This knowledge should help explain how the synthesis of the two positive-strand RNAs is regulated in infected cells.

Acknowledgments

We thank Yan Hu for technical support.

This work was funded by Public Health Service grant AI-070728.

Footnotes

[down-pointing small open triangle]Published ahead of print on 6 January 2010.

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